Biology subjects

Yirmiya, E.

Publications and source records attributed to Yirmiya, E..

15 recordsLinked to original sources

bioRxiv2026

Conserved catalytic activity of immune TIR domains in animals

The Toll/interleukin-1 receptor (TIR) domain is important for immune signaling across bacteria, plants, and animals. In human innate immunity, TIR domains are known to function as adaptors mediating protein-protein interactions, yet studies in bacteria and plants revealed that TIR domains often act as enzymes that produce immune signaling molecules. Here, we show that TIR domains from evolutionarily diverse animals have conserved active sites, implying that they can function as enzymes. In vitro experiments with animal TIRs show that the TIR domain of several Toll-like receptors (TLRs), including that of human TLR4, can produce cyclic ADP-ribose (cADPR), revealing an enzymatic activity previously unknown for TLR TIRs. We show that production of cADPR is a conserved feature of TIR domains across the animal tree of life, implying a role for this molecule in animal TIR signaling. Finally, we report a TIR domain from green algae that synthesizes 3'cADPR, suggesting conservation of 3'cADPR signaling between bacteria and eukaryotes. Our results reveal that the catalytic activity of TIR domains is widespread in animals and conserved across the tree of life.

immunology↗

bioRxiv2026

Bacterial defense via RES-mediated NAD+ depletion is countered by phage phosphatases

Many bacterial defense systems restrict phage infection by breaking the molecule NAD+ to its constituents, adenosine diphosphate ribose (ADPR) and nicotinamide (Nam). To counter NAD+ depletion-mediated defense, phages evolved NAD+ reconstitution pathway 1 (NARP1), which uses ADPR and Nam to rebuild NAD+. Here we report a bacterial defense system called aRES, involving RES-domain proteins that degrade NAD+ into Nam and ADPR-1-phosphate (ADPR-1P). This molecule cannot serve as a substrate for NARP1, so that NAD+ depletion by aRES defends against phages even if they encode NARP1. We further discover that some phages evolved an extended NARP1 pathway capable of overcoming aRES defense. In these phages, the NARP1 operon also includes a specialized phosphatase, which dephosphorylates ADPR-1P to form ADPR, a substrate from which NARP1 then reconstitutes NAD+. Other phages encode inhibitors that directly bind aRES proteins and physically block their active sites. Our study describes new layers in the NAD+-centric arms race between bacteria and phages and highlights the centrality of the NAD+ pool in cellular battles between viruses and their hosts.

microbiology↗

bioRxiv2025

Systematic discovery of TIR-based immune signaling systems in bacteria

Toll/interleukin-1 receptor (TIR) domains are important for immune signaling across humans, plants and bacteria. These domains were recently found to produce immune signaling molecules in plant immunity as well as in a family of bacterial defense systems called Thoeris. Here, we systematically scanned bacterial defense islands to identify anti-phage defense systems involving TIR-mediated signaling. We detected numerous configurations of such systems in bacterial genomes, involving [~]30 different protein effectors predicted to respond to TIR-produced immune signals. We experimentally verified 15 new TIR-containing systems, showing that they provide defense against phages through effector protein domains not previously known as associated with immunity. Further biochemical analyses revealed bacterial Thoeris systems that generate 2'cADPR, an immune signaling molecule central to plant immunity. We also discover multiple types of Thoeris that drive antiphage defense via canonical cADPR, a signaling molecule known to mediate human innate immunity. Our studies show that TIR-based immune signaling systems exist in at least 8% of bacterial genomes, and suggest conservation of TIR-derived immune signals across the tree of life.

microbiology↗

bioRxiv2025

Bacteria sense virus-induced genome degradation via methylated mononucleotides

Phages often degrade the genome of their bacterial host to individual nucleotides and use these nucleotides to build their own genome. In this study, we describe a bacterial defense system that directly senses phage-mediated host genome degradation. This system, called Metis, aborts phage infection once it detects the accumulation of the modified mono-nucleotide N-methyl-deoxyadenosine monophosphate (mdAMP). As methylation of deoxy adenosines occurs only in the context of the DNA polymer, intracellular accumulation of mdAMP serves as a definitive signal that the host genome has been degraded to its individual constituents. In type I Metis, sensing of mdAMP activates an NAD+ diphosphatase, leading to rapid NAD depletion and cessation of the infection process; while the effector in type II Metis is a transmembrane-spanning protein whose toxicity is triggered in response to the modified mono-nucleotide. We further show that Metis defense depends on endogenous DNA methylases, and that phages can escape Metis via mutations that inactivate phage-mediated host genome degradation. Our results demonstrate how molecular byproducts released during virus-induced cell exploitation can be used as specific danger signals that trigger host immunity.

microbiology↗

bioRxiv2025

Functional diversity of phage sponge proteins that sequester host immune signals

Multiple bacterial immune systems, including CBASS, Thoeris, and Pycsar, employ signaling molecules that activate the immune response following phage infection. Phages counteract bacterial immune signaling using sponge proteins that bind and sequester the immune signals, but the breadth of immune signals targeted by phage sponges is unclear. Here we study the functional versatility of Acb2, Tad1 and Tad2, three families of sponge proteins known to inhibit CBASS and Thoeris signaling. Eighty-four proteins representing the phylogenetic diversity of these sponge families were tested for their ability to inhibit immunity by sequestering 3'3'-cGAMP and 3'3'-cUA (CBASS), cCMP and cUMP (Pycsar), and 3'cADPR, His-ADPR and N7-cADPR (types I, II and IV Thoeris, respectively). While Acb2 proteins were so far reported to inhibit only CBASS systems, we found Acb2 homologs that bind 3'cADPR and inhibit Thoeris defense. In addition, we discovered sponge proteins that inhibit Pycsar and type IV Thoeris by binding cUMP and N7-cADPR, respectively. Using crystal structures, structural modeling and biochemical analyses, we explain the molecular basis for signal-binding specificities in members of these sponge families. Our study reports the first sponges inhibiting Pycsar and type IV Thoeris, and demonstrates how phage sponges evolve to obtain diverse specificities.

microbiology↗

bioRxiv2025

Structural modeling reveals viral proteins that manipulate host immune signaling

Immune pathways that use intracellular nucleotide signaling are common in animals, plants and bacteria. Viruses can inhibit nucleotide immune signaling by producing proteins that sequester or cleave the immune signals. Here we analyzed evolutionarily unrelated signal-sequestering viral proteins, finding that they share structural and biophysical traits in their genetic organization, ternary structures and binding pocket properties. Based on these traits we developed a structure-guided computational pipeline that can sift through large phage genome databases to unbiasedly predict phage proteins that manipulate bacterial immune signaling. Numerous previously uncharacterized proteins, grouped into three families, were verified to inhibit the bacterial Thoeris and CBASS signaling systems. Proteins of the Sequestin and Lockin families bind and sequester the TIR-produced signaling molecules 3'cADPR and His-ADPR, while proteins of the Acb5 family cleave and inactivate 3'3'-cGAMP and related molecules. X-ray crystallography and structural modeling, combined with mutational analyses, explain the structural basis for sequestration or cleavage of the immune signals. Thousands of these signal-manipulating proteins were detected in phage protein databases, with some instances present in well-studied model phages such as T2, T4 and T6. Our study explains how phages commonly evade bacterial immune signaling, and offers a structure-guided analytical approach for discovery of viral immune-manipulating proteins in any database of choice.

microbiology↗

bioRxiv2024

Phages reconstitute NAD+ to counter bacterial immunity

Bacteria defend against phage infection via a variety of antiphage defense systems. Many defense systems were recently shown to deplete cellular nicotinamide adenine dinucleotide (NAD+) in response to infection, by breaking NAD+ to ADP-ribose (ADPR) and nicotinamide. It was demonstrated that NAD+ depletion during infection deprives the phage from this essential molecule and impedes phage replication. Here we show that a substantial fraction of phages possess enzymatic pathways allowing reconstitution of NAD+ from its degradation products in infected cells. We describe NAD+ reconstitution pathway 1 (NARP1), a two-step pathway in which one enzyme phosphorylates ADPR to generate ADPR-pyrophosphate (ADPR-PP), and the second enzyme conjugates ADPR- PP and nicotinamide to generate NAD+. Phages encoding the NARP1 pathway can overcome a diverse set of defense systems, including Thoeris, DSR1, DSR2, SIR2-HerA, and SEFIR, all of which deplete NAD+ as part of their defensive mechanism. Phylogenetic analyses show that NARP1 is primarily encoded on phage genomes, suggesting a phage- specific function in countering bacterial defenses. A second pathway, NARP2, allows phages to overcome bacterial defenses by building NAD+ via metabolites different than ADPR-PP. Our findings report a unique immune evasion strategy where viruses rebuild molecules depleted by defense systems, thus overcoming host immunity.

microbiology↗

bioRxiv2024

TIR domains produce histidine-ADPR conjugates as immune signaling molecules in bacteria

TIR domains are central components of pattern recognition immune proteins across all domains of life. In both bacteria and plants, TIR-domain proteins were shown to recognize pathogen invasion and then produce immune signaling molecules exclusively comprising nucleotide moieties. Here we show that the TIR domain protein of the type II Thoeris defense system in bacteria produces a unique signaling molecule comprising the amino acid histidine conjugated to ADP-ribose (His-ADPR). His-ADPR is generated in response to phage infection and activates the cognate Thoeris effector by binding a Macro domain located at the C-terminus of the effector protein. By determining the crystal structure of a ligand-bound Macro domain, we describe the structural basis for His-ADPR recognition. Our analyses furthermore reveal a family of phage proteins that bind and sequester His-ADPR signaling molecules, allowing phages to evade TIR- mediated immunity. These data demonstrate diversity in bacterial TIR signaling and reveal a new class of TIR-derived immune signaling molecules combining nucleotide and amino acid moieties.

microbiology↗

bioRxiv2023

CARD-like domains mediate anti-phage defense in bacterial gasdermin systems

Caspase recruitment domains (CARDs) and pyrin domains are important facilitators of inflammasome activity and pyroptosis. Upon pathogen recognition by NLR proteins, CARDs recruit and activate caspases, which, in turn, activate gasdermin pore forming proteins to and induce pyroptotic cell death. Here we show that CARD-like domains are present in defense systems that protect bacteria against phage. The bacterial CARD is essential for protease-mediated activation of certain bacterial gasdermins, which promote cell death once phage infection is recognized. We further show that multiple anti-phage defense systems utilize CARD-like domains to activate a variety of cell death effectors. We find that these systems are triggered by a conserved immune evasion protein that phages use to overcome the bacterial defense system RexAB, demonstrating that phage proteins inhibiting one defense system can activate another. We also detect a phage protein with a predicted CARD-like structure that can inhibit the CARD-containing bacterial gasdermin system. Our results suggest that CARD domains represent an ancient component of innate immune systems conserved from bacteria to humans, and that CARD-dependent activation of gasdermins is conserved in organisms across the tree of life.

microbiology↗

bioRxiv2023

Phages overcome bacterial immunity via diverse anti-defense proteins

It was recently shown that bacteria employ, apart from CRISPR-Cas and restriction systems, a considerable diversity of phage resistance systems, but it is largely unknown how phages cope with this multilayered bacterial immunity. Here, we analyzed groups of closely related Bacillus phages that showed differential sensitivity to bacterial defense systems, and identified multiple families of anti-defense proteins that inhibit the Gabija, Thoeris, and Hachiman systems. We show that these proteins efficiently cancel the defensive activity when co-expressed with the respective defense system or introduced into phage genomes. Homologs of these anti-defense proteins are found in hundreds of phages that infect taxonomically diverse bacterial species. We show that an anti-Gabija protein, denoted Gad1, blocks the ability of the Gabija defense complex to cleave phage-derived DNA. Our data further reveal an anti-Thoeris protein, denoted Tad2, which is a "sponge" that sequesters the immune signaling molecules produced by Thoeris TIR-domain proteins in response to phage. Our results demonstrate that phages encode an arsenal of anti-defense proteins that can disable a variety of bacterial defense mechanisms.

genetics↗

bioRxiv2023

Structural basis of Gabija anti-phage defense and viral immune evasion

Bacteria encode hundreds of diverse defense systems that protect from viral infection and inhibit phage propagation1-5. Gabija is one of the most prevalent anti-phage defense systems, occurring in >15% of all sequenced bacterial and archaeal genomes1,6,7, but the molecular basis of how Gabija defends cells from viral infection remains poorly understood. Here we use X-ray crystallography and cryo-EM to define how Gabija proteins assemble into an [~]500 kDa supramolecular complex that degrades phage DNA. Gabija protein A (GajA) is a DNA endonuclease that tetramerizes to form the core of the anti-phage defense complex. Two sets of Gabija protein B (GajB) dimers dock at opposite sides of the complex and create a 4:4 GajAB assembly that is essential for phage resistance in vivo. We show that a phage-encoded protein Gabija anti-defense 1 (Gad1) directly binds the Gabija GajAB complex and inactivates defense. A cryo-EM structure of the virally inhibited state reveals that Gad1 forms an octameric web that encases the GajAB complex and inhibits DNA recognition and cleavage. Our results reveal the structural basis of assembly of the Gabija anti-phage defense complex and define a unique mechanism of viral immune evasion.

microbiology↗

bioRxiv2023

A conserved family of immune effectors cleaves cellular ATP upon viral infection

During viral infection, cells can deploy immune strategies that deprive viruses of molecules essential for their replication. In this study, we report a family of immune effectors in bacteria which, in response to phage infection, degrade cellular ATP and dATP by cleaving the N-glycosidic bond between the adenine and sugar moieties. These ATP nucleosidase effectors are widely distributed within multiple bacterial defense systems including CBASS, prokaryotic argonautes and NLR-like proteins, and we show that degradation of (d)ATP during infection halts phage propagation and aborts infection. By analyzing homologs of the immune ATP nucleosidase domain, we discover and characterize Detocs, a new family of bacterial defense systems with a two-component phosphotransfer signaling architecture. The immune ATP nucleosidase domain is also encoded within a diverse set of eukaryotic proteins that have immune-like architectures, and we show biochemically that these eukaryotic homologs preserve the ATP nucleosidase activity. Our findings suggest that ATP and dATP degradation is a cell-autonomous innate immune strategy conserved across the tree of life.

microbiology↗

bioRxiv2022

Viruses inhibit TIR gcADPR signaling to overcome bacterial defense

The Toll/interleukin-1 receptor (TIR) domain is a key component of immune receptors that identify pathogen invasion in bacteria, plants, and animals. In the bacterial antiphage system Thoeris, as well as in plants, recognition of infection stimulates TIR domains to produce an immune signaling molecule whose molecular structure remained elusive. This molecule binds and activates the Thoeris immune effector, which then executes the immune function. We identified a large family of phage-encoded proteins, denoted here Thoeris anti-defense 1 (Tad1), that inhibit Thoeris immunity. We found that Tad1 proteins are "sponges" that bind and sequester the immune signaling molecule produced by TIR-domain proteins, thus decoupling phage sensing from immune effector activation and rendering Thoeris inactive. A high-resolution crystal structure of Tad1 bound to the signaling molecule revealed that its chemical structure is 1'-2' glycocyclic ADPR (gcADPR), a unique molecule not previously described in other biological systems. Our results define the chemical structure of a central immune signaling molecule, and reveal a new mode of action by which pathogens can suppress host immunity.

microbiology↗

bioRxiv2021

Bacterial gasdermins reveal an ancient mechanism of cell death

Gasdermin proteins form large membrane pores in human cells that release immune cytokines and induce lytic cell death. Gasdermin pore formation is triggered by caspase-mediated cleavage during inflammasome signaling and is critical for defense against pathogens and cancer. Here we discover gasdermin homologs encoded in bacteria that execute prokaryotic cell death. Structures of bacterial gasdermins reveal a conserved pore-forming domain that is stabilized in the inactive state with a buried lipid modification. We demonstrate that bacterial gasdermins are activated by dedicated caspase-like proteases that catalyze site-specific cleavage and removal of an inhibitory C-terminal peptide. Release of autoinhibition induces the assembly of >200 [A] pores that form a mesh-like structure and disrupt membrane integrity. These results demonstrate that caspase-mediated activation of gasdermins is an ancient form of regulated cell death shared between bacteria and animals.

microbiology↗

bioRxiv2021

Antiviral defense via nucleotide depletion in bacteria

DNA viruses and retroviruses need to consume large quantities of deoxynucleotides (dNTPs) when replicating within infected cells. The human antiviral factor SAMHD1 takes advantage of this vulnerability in the viral life cycle, and inhibits viral replication by degrading dNTPs into their constituent deoxynucleosides and inorganic phosphate. In this study we report that bacteria employ a similar strategy to defend against phage infection. We found a family of defensive dCTP deaminase proteins that, in response to phage infection, convert dCTP into deoxy-uracil nucleotides. A second family of phage resistance genes encode dGTPase enzymes, which degrade dGTP into phosphate-free deoxy-guanosine (dG) and are distant homologs of the human SAMHD1. Our results show that the defensive proteins completely eliminate the specific deoxynucleotide (either dCTP or dGTP) from the nucleotide pool during phage infection, thus starving the phage of an essential DNA building block and halting its replication. Both defensive genes are found in a diverse set of bacterial species and are specifically enriched in Vibrio genomes. Our study demonstrates that manipulation of the deoxynucleotide pool is a potent antiviral strategy shared by both prokaryotes and eukaryotes.

microbiology↗